Adult ; Female ; Humans ; Male ; Middle Aged ; Occupational Health ; statistics & numerical data ; Regression Analysis ; Risk Factors ; Stress, Psychological ; psychology ; Surveys and Questionnaires ; Workplace ; psychology

OBJECTIVETo verify the interaction between secreted frizzled-related protein 2 (SFRP2) and osteoblast-specific factor 2 (OSF-2).

METHODSHA-tagged OSF-2 fusion protein recombinant vector pCMV-HA-OSF-2, which could express in mammal cells was constructed, then identified by enzyme-cutting and transfected into human kidney 293 (HK293) cells with or without Myc-SFRP2 recombinant eukaryotic expression vector pCMV-HA-SFRP2. The interaction between SFRP2 and OSF-2 was detected through coimmunoprecipitation and Western blotting.

RESULTSIn electrophoresis bath, target fragment of SFRP2 coding gene with 800 bp and target gene OSF-2 with 2500 bp could be seen respectively after enzyme-cutting, which showed that pCMV-Myc-SFRP2 and pCMV-HA-OSF-2 were constructed successfully. No HA-OSF-2 expression was detected after pCMV-Myc-SFRP2 or pCMV-HA-OSF-2 transfection. Whereas, HA-OSF-2 expressed by Myc antibody immunoprecipitation after pCMV-Myc-SFRP2 and pCMV-HA-OSF-2 co-transfection.

CONCLUSIONSHA-OSF-2 recombinant vector can express in mammal cells. Interaction exists between HA-OSF-2 and SFRP2.

Cell Line ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Genetic Vectors ; Humans ; Keloid ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Recombinant Fusion Proteins ; metabolism ; Transfection

A high affinity polyclonal antibody-based enzyme linked immunosorbent assay (ELISA) was developed for the quantification of zeranol in bovine urine. On the basis of urine matrix studies, the optimized dilution factors producing insignificant matrix interference were selected as 1:5 in pretreatment. In the improved ELISA, the linear response range was between 0.02 and 1 microg/ml, and the detection limit was 0.02 microg/ml for the assay. The overall recoveries and the coefficients of variation (CVs) were in the range of 82% to approximately 127% and 3.5% to approximately 8.8%, respectively. Thirty-six bovine urine samples spiked with zeranol (ranging from 0.2 to 10 microg/ml) were detected by the ELISA and liquid chromatography (LC) method, and good correlations were obtained between the two methods (R(2)=0.9643). We conclude that this improved ELISA is suitable tool for a mass zeranol screening and can be an alternative for the conventional LC method for zeranol in bovine urine.
Animals ; Calibration ; Cattle ; Chromatography, High Pressure Liquid ; Enzyme-Linked Immunosorbent Assay ; methods ; Indicator Dilution Techniques ; Zeranol ; immunology ; urine

OBJECTIVETo investigate the effects of sodium metavanadate (SMV) on blood sugar and glucose phosphorylation in mice, and to discuss the possible mechanism of its hypoglycemic effects.

METHODSDiabetic mice (D) and control mice (V) were randomly allocated to drink SMV (0.2 mg/ml) (CV and DV groups) or NaCl (80 mmol/L) (C and V groups) respectively. The study lasted for 5 weeks. Liver glucokinase, muscle hexokinase, blood glucose and insulin were assayed at the end of each week.

RESULTSBlood glucose was higher in the diabetic groups before the administration of SMV, and the blood glucose level of group DV decreased from (18.77 +/- 1.28) to (8.94 +/- 0.94) mmol/L (P < 0.01) after oral administration of SMV for one week. While liver glucokinase increased from (1.29 +/- 0.64) to (15.36 +/- 1.57) mIU/min/mg protein and muscle hexokinase increased from (1.93 +/- 0.50) to (18.62 +/- 1.71) mIU/min/mg protein (P < 0.01) respectively. There was no continuous change of these parameters during the later weeks. No significant change of serum insulin was observed in the diabetic mice. There was a remarkable negative correlation of blood glucose level with liver glucokinase and muscle hexokinase levels.

CONCLUSIONThe hypoglycemic effects of SMV was independent of insulin level. In consideration of the close relations of the activities of liver glucokinase and muscle hexokinase with diabetes, and the improving of impaired glucose phosphorylation in diabetic mice by oral sodium metavanadate, which might be the mechanism of hypoglycemic effects of SMV.

Animals ; Blood Glucose ; metabolism ; Diabetes Mellitus, Experimental ; blood ; Female ; Glucokinase ; metabolism ; Hexokinase ; metabolism ; Hypoglycemic Agents ; pharmacology ; Insulin ; blood ; Liver ; metabolism ; Mice ; Mice, Inbred ICR ; Muscle, Skeletal ; metabolism ; Phosphorylation ; Random Allocation ; Vanadates ; pharmacology

Aged ; Alcoholic Beverages ; Humans ; Hyponatremia ; etiology ; Male ; Substance Withdrawal Syndrome ; complications

OBJECTIVETo study the effects of specific small interfering RNA (siRNA) on Smoothened (Smo) gene expression and the proliferation and apoptosis of colorectal cancer LoVo cells.

METHODSThree different siRNAs (siRNA-1, siRNA-2, and siRNA-3, respectively) were transfected into LoVo cells via cationic liposome, and the changes of Smo mRNA level were determined using semi-quantitative RT-PCR 48 h after transfection. Flow cytometry and MTT assay were performed to assess the effect of the siRNAs on the proliferation and apoptosis of LoVo cells.

RESULTSForty-eight hours after Smo siRNA-1 transfection, Smo mRNA expression in LoVo cells decreased by about 63.56%, a reduction significantly greater than that in cells transfected with the other two siRNAs. The cell proliferation decreased significantly after Smo siRNA-1 transfection in comparison with the control cells, and 48 h after transfection, significantly higher apoptosis rate was observed in Smo siRNA-1-transfected cells than in the control cells.

CONCLUSIONSpecific siRNA can significantly decrease Smo mRNA expression and inhibit the proliferation while inducing apoptosis of LoVo cells.

Apoptosis ; genetics ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; genetics ; Humans ; RNA, Small Interfering ; genetics ; Receptors, G-Protein-Coupled ; deficiency ; genetics ; Smoothened Receptor ; Time Factors ; Transfection

OBJECTIVETo investigate the expression of androgen receptor (AR) and hepatitis B virus X protein (HBx) in hepatocellular carcinoma (HCC), and analyze the relationship between AR and HBx expressions.

METHODSTumor tissues and peritumoral tissues of 83 HBV-associated HCC cases were investigated in this study. Fourteen cases of HBV-negative HCC and 13 cases of hemangioma peritumoral tissues were considered as control. AR and HBx mRNA levels were determined by quantitative fluorescence real-time RT-PCR and their protein levels were assayed by Western blot. The expression of AR and HBx proteins in tissues were examined with EnVision immunohistochemical staining. The methylation status of AR promoter was determined using methylation-specific PCR (MSP).

RESULTSBoth expression levels of AR mRNA and protein of the peritumoral tissues were significantly higher (0.17) than that of tumor tissues (0.09) in HBV-associated HCC (P < 0.01), but such a difference was not found in HBV-negative HCC (0.06 vs. 0.07, P > 0.05). The level of AR expression in peritumoral tissues was associated with tumor differentiation in HBV-associated HCC. AR mRNA and protein levels of peritumoral tissues in HBV-associated HCC were significantly higher than that in HBV-negative HCC and hemangioma (all P < 0.05). In the tumor tissues, HBV-associated HCC had significantly higher AR expression than HBV-negative HCC at mRNA level (P < 0.05), but not at protein level. Spearman rank correlation analysis showed that the AR mRNA or AR protein levels were positively correlated with HBx in both tumor and peritumoral tissues in HBV-associated HCC, but the expressions of AR and HBx were not associated with AR promoter methylation status. The relative expression levels of AR mRNA and protein in the HBV-associated peritumoral tissues were negatively correlated with tumor differentiation (r = -0.213, P < 0.05; r = -0.313, P < 0.05), the higher the AR expression, the poorer differentiation. But this correlation of AR mRNA and protein was not shown in the hepatocellular carcinoma tissues.

CONCLUSIONSHBx may enhance AR expression in HBV-associated HCC, but AR promoter demethylation maybe not been involved in its main mechanism. An increased AR expression is probably an early event during the development and progression of HBV-associated HCC, and AR expression in the peritumoral tissue is correlated with HBV-associated HCC differentiation. AR may play different roles in HBV-associated HCC and HBV-negative HCC.

Adult ; Aged ; Blotting, Western ; Carcinoma, Hepatocellular ; metabolism ; pathology ; virology ; Cell Differentiation ; DNA Methylation ; Female ; Hemangioma ; metabolism ; Hepatitis B virus ; isolation & purification ; Humans ; Immunohistochemistry ; Liver ; metabolism ; Liver Neoplasms ; metabolism ; pathology ; virology ; Male ; Middle Aged ; Promoter Regions, Genetic ; RNA, Messenger ; metabolism ; Real-Time Polymerase Chain Reaction ; Receptors, Androgen ; genetics ; metabolism ; Trans-Activators ; metabolism

By using huge primer PCR Cys86 (TGC) of PoIFN-alpha was mutated to Tyr(TAC), and the first code TGT was simultaneously changed to TGC, which is a bias code of E. coli. The expression plasmid pGEX-IFN was constructed successfully. Recombinant porcine IFN alpha, which is expressed as inclusion bodies, was about 20% of the total proteins. The inclusion body was dissolved in 8 mol/L urea and subsequently renatured by dilution in refolding buffer. In order to obtain pure protein, the renatured IFN alpha was purified by FPLC, and the cytokine activity (5200 IU/mg) was verified by inhibiting the cytopathic effect.
Escherichia coli ; genetics ; Interferon-alpha ; biosynthesis ; isolation & purification ; Mutagenesis, Site-Directed ; Protein Precursors ; biosynthesis ; isolation & purification ; Recombinant Proteins ; biosynthesis ; isolation & purification

OBJECTIVETo study the expression of Smo protein and the downstream transcription factor Gli1 protein in Sonic hedgehog signal transduction pathway in gastric carcinoma.

METHODSA tissue microarray was constructed using 85 gastric carcinoma and 25 normal gastric mucosa specimens. The expression of Smo and Gli1 proteins were detected immunohistochemically and the correlation between their expression in gastric carcinoma was analyzed.

RESULTSOnly weak expression, if any, of Smo and Gli1 proteins was detected in normal gastric mucosa, but in papillary adenocarcinoma, tubular adenocarcinoma and poorly differentiated adenocarcinoma, their expressions were significant increased as the differentiation degree was lowered. Smo protein expression in gastric carcinoma was significantly correlated with that of Gli1 protein with correlation coefficient of 0.989 (P<0.001).

CONCLUSIONThe abnormal activity of Sonic hedgehog signal transduction pathway may play an important role in the occurrence of papillary adenocarcinoma, tubular adenocarcinoma and poorly differentiated adenocarcinoma, and this abnormality is associated with Smo protein overexpression, which upregulates the expression of the downstream transcription factor Gli1 protein.

Adenocarcinoma ; metabolism ; pathology ; physiopathology ; Adult ; Aged ; Female ; Hedgehog Proteins ; physiology ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Receptors, G-Protein-Coupled ; biosynthesis ; Signal Transduction ; Smoothened Receptor ; Stomach Neoplasms ; metabolism ; pathology ; physiopathology ; Transcription Factors ; biosynthesis ; Zinc Finger Protein GLI1